Our process in understanding the phosphorylation mechanisms of neurofilament protein, (NF-H) is as follows: Analysis of in vivo phosphorylated sites of tail domain of rat NF-H showed that most of the Ser -residues in the Lys-Ser-Pro (KSP) repeats of rat NF-H are phosphorylated. The structural analysis of these repeat sequences suggests that multiple kinases are involved in their phosphorylation. One of the kinases phosphorylating KSPXKX repeats is neuronal cyclin- dependent kinase-5 (cdk5). Although Cdk5 is associated with G1 cyclins (cyclin D) in mitotic cells, the kinase activity is found only in mature neuronal cells. We demonstrated that, neuronal cdk5 activity is regulated by a protein of 67kd (P67). Peak activity correlated with the maximum levels of p67 and cdk5. p67 is neurospecific, present in both CNS and PNS neurons. It is expressed in axons of hippocampal cell cultures where it colocalized with phosphorylated NF-H (P-NF-H). In addition to its role as a putative regulator of cdk5, p67 is also a syntaxin binding protein that is thought to play a role in synaptic transmission and secretion. To further characterize the role of p67 in neural tissue, we carried out an immunoblot and immunohistochemical analysis of the developing rat postnatal cerebellum using antibodies to cdk5, p67, syntaxin and P-NF-H. The immunoblots showed that all antigens were developmentally regulated, increasing in expression from PN2 to the adult, with p67 and cdk5 showing a close temporal correlation. Immunohistochemically, however, cdk5 and P-NF-H showed strong colocalization whereas syntaxin and p67 antigens were tightly colocalized in regions undergoing vigorous synaptogenesis. In fiber bundles in the deep cerebellum, however, p67, cdk5 and P-NF-H were colocalized at several stages. The results suggest that p67 may have more than one function in different regions of the developing cerebellum. Recently, another regulator protein molecule 35kd (p35), of cdk5 has been reported but its expression is restricted to the CNS. It is not clear whether both regulator molecules are required for maximal activity, or the kinase specificity is directed to different substrates by regulators. To understand the specific roles of p67 and p35 in cdk5 regulations, we have purified large quantities of bacterially expressed cdk5, p67 and p35. A quantitative evaluation of kinase activity in the presence of p67 and or p35 and phosphorylation of different neuron specific substrates molecules with KSPXKX motifs will provide some of the answers to the above questions.